Phytochrome regulates gibberellin biosynthesis during germination of photoblastic lettuce seeds

Abstract

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3beta-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3beta-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3beta-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

Figure 1

Early 13-hydroxylation GA-biosynthetic pathway in higher plants. •, Steps catalyzed by GA 20-oxidase; ○, steps catalyzed by 3β-hydroxylase. GA₁, GA₁₇, GA₁₉, and GA₂₀ have been identified in extracts of lettuce seeds (Toyomasu et al., 1993).

Figure 2

Alignment of deduced amino acid sequences of GA 20-oxidases (A) and 3β-hydroxylases (B) from lettuce seeds with GA-biosynthetic enzymes from other plants (Ps, pea; At, Arabidopsis; and Cm, pumpkin). Dark shading indicates identical residues. The sequence data of Ls20ox1, Ls20ox2, Ls3h1, and Ls3h2 will appear in the databases with the accession numbers AB012203, AB012204, AB012205, and AB012206, respectively. Ps074 (no. X91658) (Martin et al., 1996), At2301 (no. X83379) (Phillips et al., 1995), and Cm20ox (no. X73314) (Lange et al., 1994) are GA 20-oxidases, and Ps3h (Le; no. AF001219) (Martin et al., 1997), At3h (GA4; no. L37126) (Chiang et al., 1995), and Cm3h (no. U63650) (Lange et al., 1997) are GA 3β-hydroxylases from pea, Arabidopsis, and pumpkin, respectively.

Figure 3

Southern blot of genomic DNA (10 μg/lane) from lettuce under high-stringency conditions. Temperatures for hybridization and washing were both 68°C, as described in Methods. B, Digestion by BamHI; E, digestion by EcoRI; and H, digestion by HindIII. Ls20ox2 probe cDNA was also digested by EcoRI when excised from the plasmid.

Figure 4

Gene expression of GA 20-oxidases and 3β-hydroxylases from lettuce seeds during incubation under different light conditions. Total RNA (50 μg/lane) was used for northern-blot analysis. Ls3h2 mRNA (not shown) was not detectable in any lane. DM, Mature seeds; 0 h, seeds allowed to imbibe for 3 h in the dark (just before light treatments); FR, Seeds treated with far-red light; FR/R, seeds treated with far-red/red light; FR/R/FR, seeds treated with far-red/red/far-red light. Conditions for hybridization were the same as those for Southern-blot analysis. Equal loading of RNA was checked by ethidium bromide staining of ribosomal RNAs on each gel and membrane (data not shown).

Figure 5

Model of the mechanism of germination of photoblastic lettuce seed. Evidence for this model is described in the text. Open arrows indicate conversion of substances; closed arrows indicate positive action; T-bars indicate negative regulation; question marks indicate unclear relationships; and broken lines indicate more than one process.